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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-15 to 2018-03-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name: 4-methylmorpholine 4-oxide, monohydrate
- CAS-No.: 7529-22-8
- Source and lot/batch No.of test material: 14.03.2017 14:30, PA1; supplier: Lenzing AG
- Physical appearance: liquid
- Expiration date of the lot/batch: 2017-10-14
- Purity: 50.3%
- Storage condition of test material: Kept in a refrigerator set to maintain 4 °C; protected from light

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Han Wistar rat was chosen as the animal model for this study as it is a rodent species accepted by regulatory agencies for toxicity testing.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, United Kingdom
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 204-244 g (males) and 142-198 g (females)
- Fasting period before study: Not specified
- Housing: Prior to stratification, animals were housed up to 5 per cage per sex. Following stratification, animals were housed 2 or 3 per cage by sex in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms with sterilised white wood shavings as bedding material.
- Diet (e.g. ad libitum): ad libitum, SDS Rat and Mouse (modified) No. 1 Diet SQC Expanded
- Water (e.g. ad libitum): ad libitum, water from the public supply
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 43-71
- Air changes (per hr): ≥10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected for this study as it is the default route for this type of study and no marked route specificity is expected. The doses were given using a syringe with attached gavage cannula. The test and control items were administered to the appropriate animals by once daily oral gavage from Day 1 for at least 90 days. The first day of dosing as designated as Day 1 for each animal.
Vehicle:
water
Remarks:
Milli Q water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4 °C protected from light, and dispensed daily. The volume for each animal was based on the most recent body weight measurement. The required amount of test item was weighed out and the required amount of vehicle was added to achieve the required concentrations. The formulations were magnetically stirred until a homogenous formulation was obtained.

PREPARATION OF VEHICLE:
Milli-Q Water (vehicle) was dispensed weekly for administration to the control animals. An adequate amount of the vehicle was dispensed into daily aliquots, which were stored in a refrigerator set to maintain 4 °C protected from light until use.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis. The samples were obtained from the top, middle and bottom for the Group 2 to 4 preparations in study week 1, 6 and 12. All samples to be analysed were transferred immediately to the appropriate analytical laboratory at the Test Facility and were stored in a refrigerator set to maintain 4 °C until analysis or analysed on receipt.

A summary of the data from 3 analytical occasions is presented in Table 1 below. The analysed concentrations of Methylmorpholine oxide were within or equal to the acceptance criteria of ±10% of their theoretical concentration with each individual sample concentration within or equal to ± 15% of their theoretical concentration. For homogeneity, the relative standard deviation of concentrations for all samples in each group was within the acceptance criteria of ≤ 10%. No test item (Methylmorpholine oxide) was detected in the control (Group 1) formulations.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Adjusted dose of 59.6 mg/kg bw/day (when corrected for purity)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Adjusted dose of 199 mg/kg bw/day (when corrected for purity)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Adjusted dose of 596 mg/kg bw/day (when corrected for purity)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected in consultation with the Sponsor based on information from a previous study. The decision was made for 300 mg/kg bw/day to be the high dose level, with low and intermediate dose levels of 30 and 100 mg/kg bw/day selected in order to assess the dose response relationship. 30 mg/kg bw/day was anticipated to be a clear NOEL (No Observed Effect Level).
- Animals were assigned to groups by a stratified randomisation scheme designed to achieve similar group mean body weights. Males and females were randomised separately. During the week before the commencement of dosing, individual body weights were checked to ensure all animals were within ± 20% of the mean weight of each sex.
- Cages were racked by treatment group and vertically throughout the rack. Control animals were housed on a separate rack. Treated groups were housed on the same rack as required.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Animals were observed from the cage side once daily from Week -1 during the pretreatment period. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
Animals were removed from their cages and subject to detailed clinical observations weekly from Week -1 until the completion of the in-life period. Animals were observed more frequently as required for welfare reasons.

BODY WEIGHT: Yes
- Time schedule for examinations: Once prior to treatment and weekly during treatment period. Body weight was also recorded on first day of scheduled necropsy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly starting one week before treatment until completion of in-life period
- Estimated mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Monitored on a regular basis throughout the study

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once prior to treatment and once on week 13
- Dose groups that were examined: All animals prior to treatment and control as well as high dose (300 mg/kg/day) on week 13
The eyes were examined using an indirect ophthalmoscope after the application of a mydriatic agent (1% Tropicamide, Mydriacyl).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Immediately before euthanasia
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: all animals
- Parameters checked in Table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Immediately before euthanasia
- Animals fasted: No
- How many animals: all animals
- Parameters checked in Table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13 of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in Table 4 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once prior to treatment and once during week 12
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity (pain perception) / grip strength / motor activity / motor function (landing foot splay)

IMMUNOLOGY: No

OTHER:
Postdose observations
Animals were examined regularly throughout each dosing day for reaction to treatment, with particular attention being paid to the animals during and for the first hour after dosing. The onset, intensity and duration of any signs were recorded (as appropriate).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 5)
HISTOPATHOLOGY: Yes (see Table 6)
Other examinations:
Coagulation parameters (e.g activated partial thromboplastin time, prothrombin time, fibrinogen and sample quality) were also assessed from plasma.
Statistics:
All results presented in the tables of the report were calculated using non-rounded values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and have been reported at the 0.1%, 1%, and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analysed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation have been reported whenever possible.
Inferential statistics were performed according to Table 7 but excluded semi-quantitative data and any group with less than 3 observations. Levene’s test was used to assess the homogeneity of group variances. Datasets with at least 3 groups were compared using an overall one-way ANOVA F test if Levene’s test was not significant or the Kruskal-Wallis test if it was. If the overall F test or Kruskal-Wallis test was found to be significant, then the pairwise comparisons (i.e. Low Dose vs. Control, Intermediate Dose vs. Control, High Dose vs. Control) were conducted using Dunnett’s or Dunn’s test, respectively.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse treatment-related clinical observations evident in animals dosed with Methylmorpholine oxide up to 300 mg/kg bw/day.

Although increased activity was observed in 2/10 females dosed at 100 mg/kg bw/day (single occasion) and in up to 3/10 females dosed with Methylmorpholine oxide at 300 mg/kg bw/day (Days 8, 11 and 12), the incidences on each occasion were transient or isolated incidents and were therefore considered to be unrelated to treatment with Methylmorpholine oxide. In addition, one female dosed at 300 mg/kg bw/day was observed eating bedding material on one occasion (Day 31) while also convulsing (non-sustained) on Day 39. These signs were also considered unrelated to treatment due to the isolated nature of the incidents and the lack of other clinical signs recorded for this animal throughout the duration of 90 days treatment.

All other observations were deemed to be incidental background findings and not related to the test item.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were treatment-related and dose-dependent effects on body weight as well as body weight gains in both males and females receiving Methylmorpholine oxide (see Tables 1 and 2 of Results).

There was a statistically significant reduction on group mean body weight in males and females dosed at 300 mg/kg bw/day (13.3% and 7.7% decrease, respectively) compared to their concurrent respective controls. Although there was an overall dose-dependent decrease, there was no statistically significant difference in group mean body weight between males or females dosed at 30 or 100 mg/kg bw/day and their concurrent respective controls.

There was also a statistically significant reduction on group mean body weight gain in females dosed at 100 mg/kg bw/day (18.0%) and in males and females dosed at 300 mg/kg bw/day (27.7% and 26.6%, respectively) compared to the respective controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no differences in food consumption following administration of test item at dose levels up to 300 mg/kg bw/day.

The quantity of food consumed for males and females dosed at 300 mg/kg bw/day was statistically significantly lower during Weeks 1 and 2 for males and Week 1 for females when compared to the concurrent controls. For the remainder of the treatment period, food consumption remained slightly low for the males while remained similar among the other treated groups for females from approximately Day 22.

Although an initial statistical significance was achieved, a review of the pre-treatment food consumption data indicated that there is no evidence of a treatment-related effect.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in haematology or coagulation parameters at dose levels up to 300 mg/kg bw/day considered to be related to the administration of the test item. There were minor group mean differences that achieved statistical significance which were considered un-related to administration. These included statistically significantly lower red blood cell count, with high reticulocyte counts, in males dosed at 300 mg/kg bw/day and low white blood cell and lymphocyte counts, with higher platelets, in females dosed at 300 mg/kg bw/day.

A review of the individual data indicated that for the changes listed above the individual values were generally within the range of concurrent control group data for both males and females. As such, these differences were judged to be due to individual changes, biological variation or lacked a true dose relationship and were therefore considered not to be related to Methylmorpholine oxide.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
See Table 3 (for males) and Table 4 (for females) of Results for the key data regarding clinical biochemistry assessment.

There were no changes in the clinical chemistry parameters at dose levels up to 30 mg/kg bw/day considered to be related to the administration of Methylmorpholine oxide.

A dose-related decreasing trend was observed in triglycerides for both males and females, achieving statistical significance only in females dosed at 100 or 300 mg/kg bw/day when compared to concurrent controls. Additionally, globulin levels were lower for both males and females in all treated groups, achieving statistical significance for females dosed at 100 or 300 mg/kg bw/day and in males dosed at 300 mg/kg bw/day. As there were no correlating histopathological changes observed in the liver of any treated animals, the toxicological significance of these changes is unclear.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related differences in the quantitative functional observation parameters or motor activity following administration of Methylmorpholine oxide at dose levels up to 300 mg/kg bw/day.

It should be noted that while there were occasional changes in motor activity observed over a 5-minute interval and some of the changes attained statistical significance, these changes were isolated. In addition, there was a statistically significant change in the overall session (60 minutes) for basic and fine movements in males dosed at 300 mg/kg bw/day, but at least 8 out of the 10 of the individual values were within the control ranges. Therefore, these changes were considered to be incidental and not related to the administration of Methylmorpholine oxide.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg bw/day, organ weights relative to body weight were higher in the liver of both males and females and in the kidney, lung, spleen and testis of males. There were individual organ weight values that were different from their respective controls. There were, however, no patterns or correlating data (taking into account differences in sexual maturity) to suggest these values were test item-related.
Gross pathological findings:
no effects observed
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All histopathological findings observed were of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals; therefore, the histopathological findings were considered not to be test item-related.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All microscopic findings observed were of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals; therefore, the microscopic findings were considered not to be test item-related.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
other: body weight reduction
Organ:
other: body weight reduction
Treatment related:
yes
Dose response relationship:
yes

Any other information on results incl. tables

Table 1: Summary of Measured Body Weight and Body Weight Gains of Males after 90-Day Administration of Methylmorpholine Oxide

Group N Mean body weight at Day 91 [g] Standard deviation of body weight

% decrease of body weight from control (Group 1)

Mean body weight gain

(Day 1-91) [g]

Standard deviation of body weight gain

% decrease of body weight gain from control (Group 1)

1 (Control)

10

442.5

37.5

 

215.7

 33.1

2 (30 mg/kg/day)

10

423.7

36.0

 4.2

199.9

 32.1

 7.3

3 (100 mg/kg/day)

10

419.4

35.8

 5.2

194.3  29.0  9.9
4 (300 mg/kg/day) 10 383.8a 20.5  13.3  156.0a  13.1  27.7

a p0.001 compared to control (Dunnett's test).

Table 2: Summary of Measured Body Weight and Body Weight Gains of Females after 90-Day Administration of Methylmorpholine Oxide

Group N Mean body weight at Day 91 [g] Standard deviation of body weight

% decrease of body weight from control (Group 1)

Mean body weight gain

(Day 1 -91) [g]

Standard deviation of body weight gain

% decrease of body weight gain from control (Group 1)

1 (Control)

10

241.0

14.3

 

77.9 

 9.2

 

2 (30 mg/kg/day)

10

236.2

10.3

 2.0

71.2

 8.6

 8.6

3 (100 mg/kg/day)

10

228.9

25.8

 5.0

63.9b

 11.2

 18.0

4 (300 mg/kg/day)

10

222.5a

15.9

 7.7

57.2c

 7.2

 26.6

a p0.05; b p0.01; c p0.001 compared to control (Dunnett's test).

Table 3: Summary of Key Clinical Biochemistry Values (triglycerides and globulin levels) of Males after 90-Day Administration of Methylmorpholine Oxide

Group N Mean triglyceride levels [mmol/L] Standard deviation of triglyceride levels

Fold change in triglyceride levels compared to control (Group 1)

Mean globulin levels [g/L]

Standard deviation of globulin levels

Fold change in globulin levels compared to control (Group 1)

1 (Control)

10

1.955

0.917

 

18.2 

1.4

 

2 (30 mg/kg/day)

10

1.783

0.191

0.912

17.6

2.0

1.0

3 (100 mg/kg/day)

10

1.635

0.945

0.836

17.8

2.5

1.0

4 (300 mg/kg/day)

10

1.286

0.434

0.658

15.2a

1.3

0.8

a p0.01 compared to control (Dunn's test).

Table 4: Summary of Key Clinical Biochemistry Values (triglycerides and globulin levels) of Females after 90-Day Administration of Methylmorpholine Oxide

Group N Mean triglyceride levels [mmol/L] Standard deviation of triglyceride levels

Fold change in triglyceride levels compared to control (Group 1)

Mean globulin levels

[g/L]

Standard deviation of globulin levels

Fold change in globulin levels compared to control (Group 1)

1 (Control)

10

1.640

0.715

 

15.6 

1.4

 

2 (30 mg/kg/day)

10

1.315

0.573

0.802

14.7

1.5

0.9

3 (100 mg/kg/day)

10

0.968a

0.234

0.590

13.8a

1.4

0.9

4 (300 mg/kg/day)

10

0.844b

0.362

0.515

13.4b

1.4

0.9

a p0.05; b p0.01 compared to control (Dunnett's test).

Applicant's summary and conclusion

Conclusions:
In a 90-day subchronic toxicity study (OECD 408), Methylmorpholine oxide (50.3% purity) was administered orally via gavage to 10 Han Wistar rats/sex/group at dose levels of 0, 30, 100 and 300 mg/kg bw/day. Based on the results of this study, the no observed adverse effect level (NOAEL) was considered to be the intermediate dose (100 mg/kg bw/day) based on reduction in body weight (>10%) in male rats.
However, it is debatable if this effect of reduced body weight of >10% observed only in one sex (i.e. males) without corresponding effects in organ weights or histopathology is sufficient to be considered adverse. The study director recommended to not consider this an adverse effect and to stay with a NOAEL of 300 mg/kg bw/day. Nevertheless, the decision in favour of a NOAEL of 100 mg/kg bw/day is based on precaution.
Executive summary:

In a 90-day subchronic toxicity study (OECD 408), Methylmorpholine oxide (50.3% purity) was administered orally via gavage to 10 Han Wistar rats/sex/group at nominal dose levels of 0, 30, 100 and 300 mg/kg bw/day. This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral toxicity study according to OECD 408 in rats. 

There were no unscheduled deaths or treatment-related clinical observations noted. There was a treatment-related and dose-dependent effect on body weights in males and females receiving 300 mg/kg bw/day such that significantly lowered body weight by the end of the 90-day dosing period were observed in both sexes when compared to their concurrent respective controls. In particular, males dosed with 300 mg/kg bw/day had a 13.3% decrease in group mean body weight compared to the concurrent controls, which, without further observation of a recovery period (e.g. to determine if this effect is transient), can be considered as an adverse effect (e.g. body weight reduction of >10%). Females dosed with 300 mg/kg bw/day had a 7.7% decrease in group mean body weight compared to the concurrent controls. In addition to effects on body weight, there were some dose-related effects on a few clinical chemistry parameters (e.g. decreased triglycerides and globulin levels in females dosed at 100 or 300 mg/kg bw/day and in males dosed at 300 mg/kg bw/day), but as there were no correlating histopathological changes observed in the liver of any treated animals, the effects were not considered adverse.

There were no significant changes in food consumption, ophthalmology, detailed functional observations and functional tests, clinical pathology parameters, gross necropsy findings, organ weights, organ weights relative to body weight or histopathology considered to be related to treatment.

Altogether, considering the 13.3% decrease in group mean body weight of male Han Wistar rats dosed with 300 mg/kg bw/day as an adverse effect in this study, the no observed adverse effect level (NOAEL) following daily administration of Methylmorpholine oxide by oral gavage for at least 90 days was considered to be 100 mg/kg bw/day.